Neural calcium-activated neutral proteinase inhibitors

ABSTRACT

This invention is directed to two highly purified neural calcium-activated neutral proteinase (CANP or calpain) inhibitors, known as high molecular weight calpastatin (HMWC) and low molecular weight calpastatin (LMWC). The invention also relates to recombinant DNA molecules which code for, and antibodies which bind to these proteins. The present invention is further directed to the use of these calpastatin proteins.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional, of application Ser. No. 07/735,611,filed Jul. 25, 1991 now U.S. Pat. No. 5,340,922 is acontinuation-in-part of application Ser. No. 07/356,458, filed in theU.S. Patent and Trademark Office on May 25, 1989, now abandoned, whichis a continuation-in-part of application Ser. No. 07/200,141, filed inthe U.S. Patent and Trademark Office on May 31, 1988, now abandoned. Thecontents of the parent application (Ser. No. 07/200,141) andcontinuation-in-part application (Ser. No. 07/356,458) are incorporatedby reference herein.

FIELD OF THE INVENTION

This invention is directed to two highly purified neuralcalcium-activated neutral proteinase (CANP or calpain) inhibitors, knownas high molecular weight calpastatin (HMWC) low molecular weightcalpastatin (LMWC). The invention also relates to recombinant DNAmolecules which code for, and antibodies which recognize and bind to,these proteins. The present invention is further directed to the usesfor these calpastatin proteins.

BACKGROUND OF THE INVENTION

Proteolysis has been implicated not only in protein turnover, but alsoin the regulation of other physiological functions, such as, proteintranslocation, fibrinolysis, digestion, hormone maturation, andfertilization (see, e.g., Protein Degradation in Health and Disease in:CIBA Foundation Symposium 75, published by Excerpta Medica, NY (1980)).Responsible for such functions are proteinases, which enzymaticallycatalyze the degradation of protein substrates.

A number of diverse mechanisms exist which play important roles in theregulation of proteolytic activity. Particularly important mechanismsinclude modulation of proteinase activity, modulation of substrates,localization of proteinases, and sequestration of proteinases intovesicles (see, e.g., Protein Degradation in Health and Disease in: CIBAFoundation Symposium 75, published by Excerpta Medica, NY (1980)).

Calcium-activated neutral proteinase (CANP, or calpain), a non-lysosomalcysteine proteinase, is believed to participate in various intracellularprocesses mediated by calcium (Waxman, L., Methods Enzymol. 80:644-680(1981); Imahori, K., In: Calcium and Cell Function, Vol. 3, pp. 473-487(W. Y. Cheung, ed.), Academic Press, N.Y. (1982); Murachi, TrendsBiochem. Sci. 8:167-169 (1983); Mellgren, R., FASEB J. 1:110-115 (1987);Schollmeyer, J. E., Science 240:911-913 (1988); Watanake et al. Nature342:505-511 (1989)). CANPs comprise a family of enzymes that are widelydistributed in mammalian and avian cells (Murachi et al., Trends inBiochem. Sciences 8:167-169 (1983)).

The potential activity of CANPs in cells appears to greatly exceed thephysiological needs. For example, when maximally expressed underexperimental conditions, CANPs in retinal ganglion cells can hydrolyzemore than 50% of the entire content of axonal proteins within 5-15minutes (Nixon et al., J. Neurosci. 6:1252-1263 (1986)). Given thisenormous potential activity, it is not surprising that variousmechanisms exist to regulate calcium-dependent proteolysis in vivo,including cellular calcium levels, modulation of the enzyme'ssensitivity to calcium, autolytic activation, and its interaction withendogenous activating and inhibitory factors.

The role of CANPs in mediating cellular responses to the calcium signalis becoming widely accepted (Suzuki et al., FEBS Lett. 220:271-277(1987)). The evidence linking calcium-activated proteolysis to keyregulatory processes is of particular interest. The work of Pontremoliet al., PNAS USA 84:3604-3608 (1987), for example, has provided directevidence in intact cells for a role of CANP in the modulation ofresponses to external stimuli mediated by protein kinase C.

CANPs have been implicated in neurobiological phenomena ranging fromtransmembrane signaling and synaptic plasticity to disease-relatedneuronal degeneration and cell death. This suspected functionaldiversity reflects the existence of multiple CANPs and a complexregulation which includes changes in calcium concentration, interactionwith specific endogenous inhibitors (calpastatins), autolytic conversionof pro-forms to more active enzymatic forms, ability of the protease totranslocate to different cellular sites, and influences of proteinsubstrate properties.

The ability of CANPs to modulate protein kinase C and other proteinkinases suggests that these proteinases are an important component ofthe regulatory system mediating the transduction of extracellularcalcium signals. The ability of CANP to carry out limited proteolyticcleavage and thereby activate certain enzymes, including protein kinaseC, emphasizes the potential creative functions of proteolysis andimplies that the actions of CANP may be amplified intracellularly inprovocative ways.

Because CANPs are particularly well-represented in neurons (Nixon etal., J. Neurochem. 6:1252-1263 (1986); Nixon, R. A., J. Neurochem.6:1264-1271 (1986); Hamakabu et al., J. Neurosci. 6:3103-3111 (1986)),various neurobiological roles have been proposed for them, includingsynaptic modulation (Lynch et al., Science 224:1057-1063 (1984)), thepost-translational modification and degradation of cytoskeletal proteins(reviews: Nixon, R. A., In: Neurofilaments (Marotta, C. A., eds.), pp.117-154, Univ. Minnesota Press, Minneapolis, Minn. (1983); Schlaepfer,in: Neurofilaments, pp. 57-85, C. A. Marotta (ed.), Univ. of MinnesotaPress (1983)) and the modification of membrane receptors (Baudry et al.,Science 212:937-938 (1981)). CANPs appear to be involved in thepostsynaptic events of long-term potentiation (Staubli et al., BrainRes. 444:153-158 (1988)), and the mounting evidence for a regulatoryrole of CANPs in protein kinase C modulation (Suzuki et al., FEBS Lett.220:271-277 (1987)) is consistent with a role in presynaptic mechanismsof long-term potentiation (Akers et al., Science 231:587-589 (1986)).

Although the involvement of proteolytic systems in cell death is clearlyestablished, there has been a traditional bias toward regardingproteolysis as an end-stage phenomenon or as merely a scavengingmechanism for cellular debris. This traditional role for proteolyticenzymes has been revised by a new appreciation of the varied regulatoryroles of proteolytic enzymes in nearly every aspect of cellularfunction, including neurophysiological function.

Increased interest in the role of excitatoxins in mechanisms of celldeath in neurodegenerative disease and related disorders (Spencer etal., Science 237:517-519 (1987)) also implicates CANPs. Rapid influx ofcalcium can lead directly to an activation of latent CANP. Some recentstudies demonstrate that excitatoxins activate CANP and inducestructural protein breakdown in vivo (Noszek, et al., Soc. Neurosci.Abstr. 13:1684 (1987)) and that CANP inhibitors reduced the damageproduced by kainate, NMDA and quisqualate (Siman et al., Soc. Neurosci.Abstr. 13:1684 (1987)).

Such considerations are especially pertinent to an examination ofAlzheimer's Disease pathogenesis, for example. It has been found thatthe inactivation of CANP with a specific monoclonal antibody inhibitsthe conversion of membrane-bound protein kinase C to a solublecalcium-independent form, thereby increasing the production ofsuperoxides and stimulating the phosphorylation of membrane proteins.These effects are directly relevant to known mechanisms of cell deathsuch as free radical-induced cellular damage, secondarily increased Ca²⁺influx (Cross et al., Ann. Int. Med. 107:526-545 (1987)), and thestructural abnormalities found in Alzheimer's Disease brain such asaltered phosphorylation of cytoskeleton proteins (tau) and membranedamage leading to altered amyloid precursor protein processing (Dyrks etal., EMBO J. 7:949-957 (1988)).

The conspicuous susceptibility of cytoskeletal proteins to CANPs and therelative enrichment of CANPs in neurons has focused particular attentionon these proteases as regulators of neuronal cytoskeleton dynamics.

By what mechanisms CANP activity may become down-regulated and therebygive rise to intracellular accumulations of cytoskeletal proteins orprotein fragments in affected Alzheimer's Disease neurons, and at whatstage calcium influx ultimately increases to activate latent CANPs tocause irreversible cell death, remain unanswered questions ofconsiderable importance to Alzheimer's Disease and other late-onsetneurodegenerative disorders. That is why the regulators of CANPactivity, such as endogenous inhibitors, are so important.

Calpastatins, the specific protein inhibitors of CANP, are also widelydistributed among tissues. First identified in 1978 (Waxman et al., J.Biol. Chem. 253:5888-5891 (1978)), calpastatins have since been purifiedfrom several different sources. Although each of the purified speciesshares the properties of heat stability and strict specificity for CANP,there is no consensus on the number of forms of calpastatin withinsingle cells or among different cell types. The recent characterizationof a calpastatin cDNA isolated from a rabbit cDNA library (Emori et al.,Proc. Natl. Acad. Sci. USA 84:3590-3594 (1987)) revealed a deducedsequence of 718 amino acid residues (M_(r) =76,964) containing fourconsecutive internal repeats of approximately 140 amino acid residues,each expressing inhibitory activity (Emori, et al., ibid. (1987)). Thisdeduced molecular weight is significantly lower than the molecularweight of rabbit skeletal muscle calpastatin (M_(r) =110,000),suggesting that the inhibitor migrates anomalously on SDS gels and maybe post-translationally modified.

Other studies suggest that additional molecular forms of calpastatin maybe present in tissues. Although 110 kDa calpastatin is observed inrabbit and bovine skeletal muscle (Nakamura et al., J. Biochim.98:757-765 (1985); Otsuka et al., J. Biol. Chem. 262:5839-5851 (1987)),porcine cardiac muscle (Takano et al., J. Biochem. 235:97-102 (1986))and human liver (Imajoh et al., FEBS Lett. 187:47-50 (1984)), othermolecular forms of calpastatin have also been isolated, including a 68kDa form from chick skeletal muscle (Ishiura et al., Biochem. Biophys.Acta 701:216-223 (1982)) and porcine erythrocytes (Takano et al., J.Biochem. 235:97-102 (1986)), a 50 kDa heterodimer from rabbit skeletalmuscle (Nakamura et al., J. Biochem. 96:1399-1407 (1984)) and 34 kDaforms from rabbit skeletal muscle (Takahashi-Nakamura et al., J.Biochem. 90:1583-1589 (1981)) and rat liver (Yamato et al., Biochem.Biophys. Res. Comm. 115:715-721 (1983)). The sensitivity of calpastatinto proteolysis has suggested that smaller polypeptide chains containinginhibitory activity might be derived from larger precursors duringpurification, or in vivo. Although certain of these low molecular weightcalpastatins resemble the higher molecular weight forms, theirderivation from the same gene product has not been established.

Although the activity of calpastatins in the nervous system isconsiderable, little else is known about the properties of theseproteins and how they regulate calcium-mediated proteolysis in neuralcells.

SUMMARY OF THE INVENTION

The inventor initiated studies on neural calpastatins, the calpaininhibitory factors. As part of this research, two related endogenousneural, especially human brain, calcium-activated neutral proteinase(CANP or calpain) inhibitors, known as high molecular weight calpastatin(HMWC) and low molecular weight calpastatin (LMWC) were identified andpurified.

The high molecular weight (HMW) calpastatin (HMWC) is a protein with anative molecular weight of 300 kilodaltons (kDa) which was purified tohomogeneity from postmortem human brain. The denatured molecular weightof HMWC was determined to be 41 kilodaltons. The 41 kDa polypeptide aswell as the native 300 kDa protein were heat stable and specific forcalcium-activating neutral proteinases. Antibodies raised against thepurified protein reacted selectively with the 41 kDa polypeptide, inaddition to forming an immunocomplex with the native protein. Thepurified inhibitor exhibited an isoelectric point of 4.5 (pI=4.5) bypolyacrylamide gel isoelectric focusing.

The low molecular weight (LMW) calpastatin (LMWC) identified has anative molecular weight of 60 kDa. The low molecular weight calpastatin(LMWC) has a denatured molecular weight of 31 kDa and a pI range of4.2-4.7. LMWC is heat stable and specific for calcium-sensitive forms ofCANP.

The LMWC and HMWC show a lack of immunologic relatedness. None of theantibodies raised against the HMWC cross reacted with the 60 kDa or 31kDa LMWC proteins in unfractionated brain tissue or in purified form byWestern blot analysis. Furthermore, analysis of the N-terminal sequenceof each of the peptides, i.e., 41 and 31 kDa, revealed little if anyhomology between these proteins. Although some observations suggest thatthe low molecular weight calpastatin may be proteolytic products of thehigh molecular weight form, no homology was found by protein sequencing,indicating that the calpastatin proteins of the present invention aredifferent.

The amino acid composition of HMWC and LMWC is distinct from that ofpreviously reported pig heart and rabbit liver calpastatins (Emori etal., PNAS USA 84:3590-3594 (1987) and Murachi et al., Biochem. J.84:3590(1987)).

The inhibitors of the present invention have major implications forunderstanding, diagnosing, and controlling various neurodegenerativedisorders.

DESCRIPTION OF THE FIGURES

FIG. 1. Flow diagram for the purification of high molecular weightcalpastatin from human brain. Native molecular weight: MW--300,000;reduced and denatured HMWC on SDS-PAGE: MW--41,000; isoelectric point:4.5; Immunocharacterization: (Sheep IgG: I-2-7); 2-D gelimmunoblotting--a single spot (MW 41,000,pI: 4.5); native calpastatinformed complex with I-2-7.

FIG. 2. Flow diagram for the purification of low molecular weightcalpastatin from human brain. Native molecular weight: MW--60,000;reduced and denatured LMWC on SDS-PAGE: MW 31,000; isoelectric point:pI: 4.2-4.7; two-dimensional gel electrophoresis: LMWC 31 KDa (pI4.2-4.7).

FIG. 3. Ion exchange chromatography of calpastatin from human cerebralcortex: Proteinase inhibitory activity (closed circles) and absorbanceat 280 nm (open circles). Chromatography of a 30,000 g supernatantextract of brain was carried out on DEAE-cellulose (see Examplessection). Enzyme activity was eluted with a 0-0.4M linear gradient ofKCl. Aliquots of each fraction were assayed for CANP inhibitory activityas described in the Examples section.

FIG. 4. Gel filtration chromatography of DEAE-cellulose purifiedcalpastatin: CANP inhibitory activity (closed circles) and relativeprotein concentration (open circles) determined by the method ofBradford, M. M., Anal. Biochem., 72:248-254 (1976). The active fractionsfrom FIG. 3 were concentrated by ammonium sulfate precipitation andapplied to a column of Ultrogel AcA-44 which was eluted as described inthe Examples section. The active fractions subjected to furtherpurification are indicated. Peaks of inhibitory activity correspondingto HMWC and LMWC are indicated in the figure.

FIG. 5. DEAE-Sepharose 4B chromatography of Ultrogel AcA-44 purified HMWcalpastatin: CANP inhibitory activity (solid lines) and relative proteinconcentration (broken lines). The active fractions corresponding to HMWCindicated in FIG. 4 were pooled, adjusted to 0.3M KCl and applied to aDEAE-Sepharose 4B column. HMWC activity was eluted with buffer D asdescribed in the Examples section.

FIG. 6. Gel chromatography of HMWC from human brain on Ultrogel AcA34.HMWC from DEAE-Sepharose fraction was heated at 100° C. for 5 min.Heat-treated HMWC fraction was centrifuged at 100,000×g for 30 min. Thesupernatant was recovered and applied to a column (2.5×115 cm) ofUltrogel AcA34. Five ml fractions were collected and assayed for theirability to inhibit mCANP. The elution positions of standard proteins(Ferritin, 450 kDa; Catalese, 240 kDa; aldolase, 160 kDa; and bovineserum albumin, 68 kDa) were determined in separate experiments.

FIG. 7. Two-dimensional gel electrophoresis and immunoblotting ofpurified HMWC. Purified HMWC was electrophoresed on the gels containing6.9% acrylamide, 2.5% ampholine (pH 3-8), 1.6M urea and 0.5% NP-40.After completion of 2nd-dimensional gels on SDS-PAGE (10% polyacylamidegels), proteins were electrophoretically transferred to PVDF-membranefor immuno-staining of HMWC with affinity purified I-2-7 (sheeppolyclonal antibody to HMWC). The arrow indicates the position ofpurified HMWC at pI=4.5.

FIG. 8. Isoelectric focusing of HMWC of human brain. Purified HMWC fromUltrogel AcA34 column was electrophoresed on the polyacrylamideisoelectric focusing gels in ampholine 3-8 (pH) at 4° C. Afterelectrophoresis, the gel was sliced into 3 mm segments. Each slice wasincubated with mCANP at 0° C. for 10 hours by gentle shaking.Calpastatin activity was examined by the standard protocol. The arrowindicates the position of HMWC (at pI=4.5) on isoelectric focusing gel.

FIG. 9. Effect of varying concentrations of brain calpastatin onpurified human brain mCANP. Varying amounts of purified LMWC and HMWCwere incubated with 0.43 μg of purified human brain mCANP (Vitto et al.,J. Neurochem. 47:1039-1051 (1986)) for 15 min under optimal conditionsfor CANP activity (see Examples). Relative proteolytic activity wasmeasured as the release of TCA-soluble peptides and amino acids from ¹⁴C-azocasein (see Examples). The effects of HMWC (FIG. 9A) and LMWC (FIG.9B) are shown.

FIG. 10. Immunoprecipitate of HMWC calpastatin by I-2-7-polyclonalantibody. Ten μl of the purified HMWC was incubated with various amountsof antibody (I-2-7) raised against HMWC from human brain. IgG-HMWCcomplexes were removed by Protein G. Remaining supernatants wereexamined for inhibitor activities.

FIG. 11. Ion-exchange chromatography with CM cellulose: The pooledfractions of LMWC obtained from Ultrogel AcA-44 column were purifiedover a CM-cellulose ion-exchange column at pH 5.0. LMWC was dialyzedagainst 20 mM Na-acetate, pH 5.0, 1 mM EGTA, 2 mM EDTA, 1 mM DTT, 1 mMbenzamidine at 0° C. The dialyzate was applied to a column (2.5×25 cm)of CM-cellulose (CM-52, Whatman) equilibrated with buffer E. After thecolumn was washed with 800 ml of buffer E, the column was eluted with alinear gradient of 0-0.4M NaCl (total 700 ml) in buffer E. The flow ratewas 50 ml/hr, and 10 ml fractions were collected. The inhibitorfractions were pooled and concentrated with the aid of Centriprep-10(Amicon Corp.).

FIG. 12. Gel chromatography of LMWC from human brain on Sephacryl S-300.The supernatant of heat-treated partially purified LMWC was applied toSephacryl S-300. The column was eluted with 20 mM Tris-HCl, pH 7.4,0.15M KCl, 0.5 mM DTT, 2 mM EDTA, 1 mMEDTA, 1 mM EGTA, 1 mM benzamidineand 0.004% NaN₃ and 5 ml of fractions was collected. Each fraction wasassayed for the inhibition of mCANP. The elution positions of standardproteins (Aldolase, 160 kDa; bovine serum albumin, 68 kDa; ovalbumin, 45kDa; and chymotryposinogen A, 25 kDa) were determined in separateexperiments.

FIG. 13. Isoelectric focusing of LMWC of human brain. Purified LMWC waselectrophoresed on the isoelectric focusing gels in ampholine 3-8 (pH)at 4° C. After electrophoresis, the gel was sliced into 3 mm segmentsfor inhibitor assay. Two peaks are shown with inhibitory activity by thestandard inhibitor assay protocol.

DETAILED DESCRIPTION OF THE INVENTION

A. Low and High Molecular Weight Calpastatin

Calcium-activated neutral proteinases (CANPs or calpains) are a familyof cysteine endopeptidases in vertebrate tissues. The physiologicalroles proposed for calpains include activation of enzymes (especiallyprotein kinases), modification of receptor binding properties,participation in general protein turnover, modification and degradationof cytoskeletal proteins, and regulation of membrane-cytoskeletoninteractions. In addition, calcium-activated proteolysis partly mediatesthe degeneration following axotomy and axonal injury and has beenproposed as a pathogenetic factor in certain neurodegenerativedisorders.

Endogenous protein inhibitors of calpains, called calpastatins, areheat-stable polypeptides with high specificity for calcium-dependentproteinases. Calpastatins are essential factors in the in vivoregulation of CANP activity, and perturbations of this ratio ofinhibitor to enzyme in non-neural tissues have the predictedconsequences on CANP activity in cells.

According to this invention, two forms of neural calpastatin proteinshave been identified and purified: a high molecular weight calpastatin(HMWC) and a low molecular weight calpastatin (LMWC).

The HMW calpastatin is a protein with a molecular weight of 300 kDa,with a denatured molecular weight of 41 kDa. The HMWC is heat stable andspecific for calcium-activating neutral proteinases. Antibodies raisedagainst HMWC react selectively with the 41 kDa peptide on immunoblotsafter 2-D gel protein analysis. This antibody also selectively binds tothe native 300 kDa protein, forming an immunocomplex.

The purified HMWC exhibited an isoelectric point of 4.5 bypolyacrylamide gel isoelectric focusing. The amino acid composition ofHMWC is shown in Table 3, and the N-terminal amino acid sequence (19amino acids) of HMWC 41 kDa peptide was determined as follows:

NH₂-X-Met-Pro-Pro-Glu-Pro-Ala-Thr-Leu-Lys-Gly-X-Val-Pro-Asp-Asp-Ala-Val-Glu(SEQ ID NO: 1)

wherein X represents an unknown amino acid residue.

The amino acid sequence of HMWC is different from any other calpastatinreported in the past, distinguishing this protein as a distinct andnovel form of calpastatin.

The present invention also concerns a LMWC protein. The LMWC is aheat-stable protein which is specific for calcium-sensitive forms ofCANP. The molecular weight of this protein was determined to be 60 kDa.The native protein consisted of a 31 kDa dimer with a pI range of4.2-4.7 on isoelectric focusing gels.

The amino acid composition of LMWC is disclosed in Table 6. TheN-terminal sequence of LMWC for the first 20 amino acid residues wasdetermined as follows:

NH₂-X-Glu-Lys-Glu-Thr-Lys-Glu-Glu-Gly-Lys-Pro-Lys-Gln-Gln-Gln-X-X-Lys-Glu-Lys(SEQ ID NO. 2)

wherein X represents an unknown amino acid residue.

HMWC and and LMWC show a lack of immunologic relatedness. None of theantibodies raised against the HMWC cross reacted with LMWC inunfractionated brain tissue or in purified form by Western blotanalysis. Furthermore, analysis of the N-terminal sequence of each ofthe peptides, i.e., 41 and 31 kDa, revealed little if any homologybetween these proteins.

The purified HMWC differs in molecular weight, amino acid composition,and isoelectric point from the calpastatin isolated previously fromchicken (Ishiura et al., Biochim. Biophys. Acta 701:216-223 (1982) andrabbit (Takahashi-Nakamura et al., J. Biochem. 96:1399-1407 (1981)skeletal muscle and differs in molecular weight from the inhibitorisolated from human erythrocytes (Murakami et al., J. Biochem90:1798-1816 (1981)). In addition, the calpastatin proteins of thepresent invention differ in size from a number of calpastatin proteinsisolated from other sources including bovine cardiac muscles which werereported to contain a monomeric inhibitor of 145,000 daltons (Mellgrenet al., ABB 22:779-786 (1983)). DeMartino and Croall purified a dimericinhibitor of 125,000 daltons from rat liver (ABB, 232:713-720 (1984).

The amino acid composition of the 41 kDa peptide of HMWC and the 31 kDapeptide of LMWC are distinct from that of previously reported pig heartand rabbit liver calpastatins (Emori et al., PNAS USA 84:3590-3594(1987) and Takano et al., Biochem. J. 235:97-102 (1986)).

B. Isolation of Low and High Molecular Weight Calpastatin

In accordance with this invention, LMWC and HMWC can be isolated from asample containing the enzymes. Any sample that contains the enzymes maybe used as a starting material according to the methods described inthis invention. The sample will typically be neural tissue, particularlybrain tissue and more particularly cerebral cortex tissue, and can beeither gray or white matter. The neural tissue can be from anyvertebrate source, preferably from mammalian brain and more preferablyfrom human brain. However, the calpastatins described by the presentinvention have also been identified in mouse and bovine brain, spinalcord, and murine optic nerve. The calpastatin proteins of the presentinvention may also be isolated from host cells which express therecombinant calpastatin protein(s).

The isolation and purification of calpastatin proteins of the presentinvention are described herein from a human cerebral cortex sample,although it is to be understood that other brain samples could be usedas the source material.

According to this invention, LMWC and HMWC are purified to homogeneityby a series of chromatographic steps. The proteins were isolated usingmethods that did not require exposing the inhibitors to calciumtreatment, a factor that might alter the properties of calpastatin.Although the purification procedure involved a heat treatment step, thistreatment was found not to alter the properties of these proteins.

The tissue sample according to the present invention was firsthomogenized in a buffer solution; HMWC and LMWC separated into thesupernatant fraction following centrifugation. The calpastatin proteinsof the present invention were then purified to homogeneity by a numberof biochemical purification techniques including precipitation, ionexchange, gel filtration, hydrophobic interaction, two dimensional gelelectrophoresis and isoelectric focusing gels.

The specific techniques for isolation and purification of the HMWCcalpastatin of the present invention involved anion exchange ondiethylaminoethyl (DEAE)-cellulose, gel filtration on Ultrogel AcA-44, aDEAE-Sepharose CL4B ion-exchange column, heat-treatment (100° C., 5 min)and Ultrogel AcA34 gel chromatography. Purification techniques for LMWcalpastatin, on the other hand, involved anion exchange onDEAE-cellulose, gel filtration on Ultrogel AcA44, ion exchange onCM-cellulose, affinity chromatograpahy on Con A-Sepharose, heattreatment, and Sephacryl-300 chromatography. One of skill in the artwill know of equivalents to these particular chromatographic columnswhich may be employed in isolating and purifying the proteins of thisinvention. Furthermore, as will be apparent to those of skill in theart, affinity chromatography, for example, with antibodies specific forHMWC or LMWC, may be used to purify the proteins of the presentinvention. Techniques of immunoaffinity purification are described byHarlow et al., In: Antibodies, A Laboratory Manual, Coldspring Harbor,N.Y. (1988) which is herein incorporated by reference.

Although differing slightly in their affinity for DEAE-cellulose, bothcalpastatins were not separated at this step. Gel filtrationchromatography on Ultrogel AcA 44, however, resolved two peaks ofinhibitory activity, designated high molecular weight calpastatin (HMWC)and low molecular weight calpastatin (LMWC). The purification schemesfor HMWC and LMWC are represented in FIGS. 1 and 2, respectively.

Calpastatin activity was measured as the inhibition of ¹⁴ C-azocaseindegradation by purified human brain calpain II. The HMWC and the LMWC,separated at the Ultrogel step, should each have accounted for abouthalf of the total inhibitory activity in the brain tissue. The twocalpastatins exhibited approximately the same specific activities.

In the process of this invention, HMWC was purified 964-fold while LMWCwas purified 528-fold, as measured by inhibitor activity, withapproximately 19.5% recovery in HMWC and 15% recovery in LMWC of totalactivity.

In a preferred embodiment, without being limiting, a purification schemefor low molecular weight calpastatin with a molecular weight of about 60kDa and having an activity defined as the ability to inhibit theactivity of purified calcium-activated neutral proteinase, wouldcomprise:

A. recovering crude LMWC from a neural sample;

B. subjecting said crude LMWC from step (A) to ion exchangechromatography to obtain active fractions of LMWC as defined as theability to inhibit the activity of purified calcium-activated neutralproteinase;

C. subjecting said active fractions of LMWC from step (B) to gelfiltration to obtain partially purified LMWC;

D. subjecting said partially purified LMWC from step (C) to ion exchangechromatography to obtain partially purified LMWC;

E. heat treating said partially purified LMWC from step (D); and

F. purifying said partially purified LMWC from step (E) by gelfiltration to obtain substantially pure LMWC.

Similarly, in a preferred embodiment, without being limited, apurification scheme for high molecular weight calpastatin (HMWC) with amolecular weight of about 300 kDa, said HMWC having one subunitpolypeptide with a molecular weight of about 41 kDa, having an enzymeactivity as defined as the ability to inhibit the activity of purifiedcalcium-activated neutral proteinase, would comprise:

(a) recovering crude HMWC from a neural sample;

(b) subjecting said crude HMWC from step (a) to ion exchangechromatography to obtain active fractions of HMWC by defined as theability to inhibit the activity of purified calcium-activated neutralproteinase;

(c) subjecting said active fractions of HMWC from step (b) to gelfiltration to obtain partially purified HMWC;

(d) subjecting said partially purified HMWC from step (c) to ionexchange chromatography to obtain partially purified HMWC;

(e) heat treating said partially purified HMWC from step (d);

(f) subjecting said partially purified HMWC from step (e) to gelfiltration; and

(g) obtaining substantially purified HMWC.

Using the above-described series of purification steps, calpastatin wassubstantially purified over the cell extract. As used herein, the term"substantially pure" or "substantially purified" is meant to describeHMWC or LMWC which is substantially free of any compound normallyassociated with the enzyme in its natural state, i.e., substantiallyfree of contaminating protein and carbohydrate components. The term isfurther meant to describe calpastatins of the present invention whichare homogeneous by one or more purity or homogeneity characteristicsused by those of skill in the art. For example, substantially purecalpastatin proteins will show constant and reproducible characteristicswithin standard experimental deviations for parameters such as thefollowing: molecular weight, chromatographic techniques, and such otherparameters. The term is not meant to exclude the presence of minorimpurities which do not interfere with the biological activity of theenzyme, and which may be present, for example, due to incompletepurification.

C. Cloning Calpastatin Genes

Any of a variety of procedures may be used to clone the calpastatingenes of the present invention. One such method entails analyzing ashuttle vector library of DNA inserts (derived from brain tissue whichexpresses calpastatin proteins) for the presence of an insert whichcontains the calpastatin genes. Such an analysis may be conducted bytransfecting cells with the vector and then assaying for expression ofthe calpastatin inhibitory activity. The preferred method for cloningthese genes entails determining the amino acid sequence of thecalpastatin proteins. To accomplish this task the desired calpastatinprotein may be purified and analyzed by automated sequencers.Alternatively, each protein may be fragmented as with cyanogen bromide,or with proteases such as papain, chymotrypsin or trypsin (Oike, Y. etal., J. Biol. Chem. 257:9751-9758 (1982); Liu, C. et al., Int. J. Pept.Protein Res. 21:209-215 (1983)). Although it is possible to determinethe entire amino acid sequence of these proteins, it is preferable todetermine the sequence of peptide fragments of these molecules. If thepeptides are greater than 10 amino acids long, the sequence informationis generally sufficient to permit one to clone a gene such as the genefor a particular nuclease or ligand.

The N-terminal amino acid sequence for the calpastatin proteins of thepresent invention was determined and is depicted in tables 5 and 7.

Once one or more suitable peptide fragments have been sequenced, the DNAsequences capable of encoding them are examined. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid (Watson, J. D., In: Molecular Biology of the Gene,3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif. (1977), pp. 356-357).The peptide fragments are analyzed to identify sequences of amino acidswhich may be encoded by oligonucleotides having the lowest degree ofdegeneracy. This is preferably accomplished by identifying sequencesthat contain amino acids which are encoded by only a single codon.Although occasionally such amino acid sequences may be encoded by only asingle oligonucleotide, frequently the amino acid sequence can beencoded by any of a set of similar oligonucleotides. Importantly,whereas all of the members of the set contain oligonucleotides which arecapable of encoding the peptide fragment and, thus, potentially containthe same nucleotide sequence as the gene which encodes the peptidefragment, only one member of the set contains a nucleotide sequence thatis identical to the nucleotide sequence of this gene. Because thismember is present within the set, and is capable of hybridizing to DNAeven in the presence of the other members of the set, it is possible toemploy the unfractionated set of oligonucleotides in the same manner inwhich one would employ a single oligonucleotide to clone the gene thatencodes the peptide.

In a manner exactly analogous to that described above, one may employ anoligonucleotide (or set of oligonucleotides) which have a nucleotidesequence that is complementary to the oligonucleotide sequence or set ofsequences that is capable of encoding the peptide fragment.

A suitable oligonucleotide, or set of oligonucleotides Which is capableof encoding a fragment of the desired calpastatin gene (or which iscomplementary to such an oligonucleotide, or set of oligonucleotides) isidentified (using the above-described procedure), synthesized, andhybridized, by means well known in the art, against a DNA or a cDNApreparation depending upon the source of the gene. Typically, isolationof eukaryotic genes is done by screening a cDNA library, while a DNAlibrary is used to isolate prokaryotic genes. Techniques of nucleic acidhybridization are disclosed by Maniatis, T. et al., In: MolecularCloning, a Laboratory Manual, Second Edition, Coldspring Harbor, N.Y.(1989), and by Haymes, B. D. et al., In: Nucleic Acid Hybrization, aPractical Approach, IRL Press, Washington, D.C. (1985), which referencesare herein incorporated by reference. The source of the cDNA usedaccording to the present invention will preferably be obtained byextracting RNA from human brain cells.

Techniques such as, or similar to, those described above havesuccessfully enabled the cloning of genes for streptavadin (Argarana etal., Nucleic Acids Research 14(4):1871-1882 (1986), avidin (Kulomma etal., J. Cell Biochem. Supp. part 2:210 (1988), human hepatitis type Bantibody (Hong et al., Korean J. Biochem., 18(1):7-18 (1986)), humanaldehyde dehydrogenases (Hsu, L. C. et al., Proc. Natl. Acad. Sci. USA82:3771-3775 (1985)), fibronectin (Suzuki, S. et al., Eur. Mol. Biol.Organ. J. 4:2519-2524 (1985)), the human estrogen receptor gene (Walter,P. et al., Proc. Natl. Acad. Sci. USA 82:7889-7893 (1985)), tissue-typeplasminogen activator (Pennica, D. et al., Nature 301:214-221 (1983))and human term placental alkaline phosphatase complementary DNA (Kam, W.et al., Proc. Natl. Acad. Sci. USA 82:8715-8719 (1985)).

In a alternative way of cloning calpastatin genes, a library ofexpression vectors is prepared by cloning DNA or cDNA, from a cellcapable of expressing calpastatin into an expression vector. The libraryis then screened for members capable of expressing a protein which bindsto anti-HMWC or anti-LMWC antibody, and which has a nucleotide sequencethat is capable of encoding polypeptides that have the same amino acidsequence as the calpastatin proteins of the present invention, orfragments or variants thereof.

D. Expression of Calpastatin Genes

DNA molecules composed of a calpastatin gene or at least portions ofthese genes can be operably linked into an expression vector andintroduced into a host cell to enable the expression of these proteinsby that cell. Two DNA sequences (such as a promoter region sequence anda desired calpastatin protein encoding sequence) are said to be operablylinked if the nature of the linkage between the two DNA sequences doesnot (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region sequence to direct thetranscription of the desired protein encoding gene sequence, or (3)interfere with the ability of the desired protein gene sequence to betranscribed by the promoter region sequence.

A DNA sequence encoding a calpastatin protein may be recombined withvector DNA in accordance with conventional techniques. The presentinvention encompasses the expression of the desired fusion proteins ineither prokaryotic or eukaryotic cells. Eukaryotic hosts include yeast(especially Saccharomyces), fungi (especially Aspergillus), mammaliancells (such as, for example, human or primate cells) either in vivo, orin tissue culture.

Yeast and mammalian cells provide substantial advantages in that theycan also carry out post-translational peptide modifications includingglycosylation. A number of recombinant DNA strategies exist whichutilize strong promoter sequences and high copy number of plasmids whichcan be utilized for production of the desired proteins in these hosts.

Yeasts recognize leader sequences on cloned mammalian gene products andsecrete peptides bearing leader sequences (i.e., pre-peptides).Mammalian cells provide post-translational modifications to proteinmolecules including correct folding or glycosylation at correct sites.

Mammalian cells which may be useful as hosts include cells of fibroblastorigin such as VERO or CHO-K1, and their derivatives. For a mammalianhost, several possible vector systems are available for the expressionof the desired fusion protein. A wide variety of transcriptional andtranslational regulatory sequences may be employed, depending upon thenature of the host. The transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, where the regulatory signalsare associated with a particular gene which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, etc., may be employed. Transcriptionalinitiation regulatory signals may be selected which allow for repressionor activation, so that expression of the genes can be modulated. Ofinterest are regulatory signals which are temperature-sensitive so thatby varying the temperature, expression can be repressed or initiated, orare subject to chemical regulation, e.g., metabolite.

The expression of the desired fusion protein in eukaryotic hostsrequires the use of eukaryotic regulatory regions. Such regions will, ingeneral, include a promoter region sufficient to direct the initiationof RNA synthesis. Preferred eukaryotic promoters include the promoter ofthe mouse metallothionein I gene (Hamer, D., et al., J. Mol. Appl. Gen.1:273-288 (1982)); the TK promoter of Herpes virus (McKnight, S., Cell31:355-365 (1982)); the SV40 early promoter (Benoist, C., et al., Nature(London) 290:304-310 (1981)); the yeast gal4 gene promoter (Johnston, S.A., et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975 (1982); Silver, P.A., et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at thecodon which encodes the first methionine. For this reason, it ispreferable to ensure that the linkage between a eukaryotic promoter anda DNA sequence which encodes the desired fusion protein does not containany intervening codons which are capable of encoding a methionine (i.e.,AUG). The presence of such codons results either in the formation of afusion protein (if the AUG codon is in the same reading frame as thedesired fusion protein encoding DNA sequence) or a frame-shift mutation(if the AUG codon is not in the same reading frame as the desired fusionprotein encoding sequence).

The expression of the calpastatin proteins can also be accomplished inprocaryotic cells. Preferred prokaryotic hosts include bacteria such asE. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, etc.The most preferred prokaryotic host is E. coli. Bacterial hosts ofparticular interest include E. coli K12, and other enterobacteria (suchas Salmonella typhimurium or Serratia marcescens), and variousPseudomonas species. The prokaryotic host must be compatible with thereplicon and control sequences in the expression plasmid.

To express the desired calpastatin proteins in a prokaryotic cell (suchas, for example, E. coli, B. subtilis, Pseudomonas, Streptomyces, etc.),it is necessary to operably link the desired fusion protein encodingsequence to a functional prokaryotic promoter. Such promoters may beeither constitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, and the bla promoter of the b-lactamasegene of pBR322, etc. Examples of inducible prokaryotic promoters includethe major right and left promoters of bacteriophage λ (P_(L) and P_(R)),the trp, recA, lacZ, lacI, gal, and tac promoters of E. coli, thea-amylase (Ulmanen, I., et al., J. Bacteriol. 162:176-182 (1985)), thes-28-specific promoters of B. subtilis (Gilman, M. Z., et al., Gene32:11-20 (1984)), the promoters of the bacteriophages of Bacillus(Gryczan, T. J., In: The Molecular Biology of the Bacilli, AcademicPress, Inc., NY (1982)), and Streptomyces promoters (Ward, J. M., etal., Mol. Gen. Genet. 203:468-478 (1986)). Prokaryotic promoters arereviewed by Glick, B. R., (J. Ind. Microbiol. 1:277-282 (1987));Cenatiempo, Y. (Biochimie 68:505-516 (1986)); and Gottesman, S. (Ann.Rev. Genet. 18:415-442 (1984)).

Proper expression in a prokaryotic cell also requires the presence of aribosome binding site upstream from the gene-encoding sequence. Suchribosome binding sites are disclosed, for example, by Gold, L., et al.(Ann. Rev. Microbiol. 35:365-404 (1981)).

The desired protein encoding sequence and an operably linked promotermay be introduced into a recipient prokaryotic or eukaryotic cell eitheras a non-replicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule. Sincesuch molecules are incapable of autonomous replication, the expressionof the desired receptor molecule may occur through the transientexpression of the introduced sequence. Alternatively, permanentexpression may occur through the integration of the introduced sequenceinto the host chromosome.

In one embodiment, a vector is employed which is capable of integratingthe desired gene sequences into the host cell chromosome. Cells whichhave stably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay complement an auxotrophy in the host (such as leu2, or ura3, whichare common yeast auxotrophic markers), biocide resistance, e.g.,antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene can either be directly linked to the DNA genesequences to be expressed, or introduced into the same cell byco-transfection.

In a preferred embodiment, the introduced sequence will be incorporatedinto a plasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orvital vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to "shuttle" the vector between host cells of different species.

Any of a series of yeast gene expression systems can be utilized.Examples of such expression vectors include the yeast 2-micron circle,the expression plasmids YEP13, YCP and YRP, etc., or their derivatives.Such plasmids are well known in the art (Botstein, D., et al., MiamiWntr. Symp. 19:265-274 (1982); Broach, J. R., In: The Molecular Biologyof the Yeast Saccharomyces: Life Cycle and Inheritance, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach,J. R., Cell 28:203-204 (1982)).

For a mammalian host, several possible vector systems are available forexpression. One class of vectors utilizes DNA elements which provideautonomously replicating extra-chromosomal plasmids, derived from animalviruses such as bovine papilloma virus, polyoma virus, adenovirus, orSV40 virus. A second class of vectors relies upon the integration of thedesired gene sequences into the host chromosome. Cells which have stablyintegrated the introduced DNA into their chromosomes may be selected byalso introducing one or more markers which allow selection of host cellswhich contain the expression vector. The marker may provide forprototropy to an auxotrophic host, biocide resistance, e.g.,antibiotics, or heavy metals, such as copper or the like. The selectablemarker gene can either be directly linked to the DNA sequences to beexpressed, or introduced into the same cell by co-transformation.Additional elements may also be needed for optimal synthesis of mRNA.These elements may include splice signals, as well as transcriptionpromoters, enhancers, and termination signals. The cDNA expressionvectors incorporating such elements include those described by Okayama,H., Mol. Cell. Biol. 3:280 (1983), and others.

Preferred prokaryotic vectors include plasmids such as those capable ofreplication in E. coli such as, for example, pBR322, ColE1, pSC101,pACYC 184, πVX. Such plasmids are, for example, disclosed by Maniatis,T., et al. (In: Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (1982)). Bacillus plasmidsinclude pC194, pC221, pT127, etc. Such plasmids are disclosed byGryczan, T. (In: The Molecular Biology of the Bacilli, Academic Press,NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pIJ101(Kendall, K. J., et al., J. Bacteriol. 169:4177-4183 (1987)), andStreptomyces bacteriophages such as φC31 (Chater, K. F., et al., In:Sixth International Symposium on Actinomycetales Biology, AkademiaiKaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids arereviewed by John, J. F., et al. (Rev. Infect. Dis. 8:693-704 (1986)),and Izaki, K. (Jpn. J. Bacteriol. 33:729-742 (1978)).

Once the vector or DNA sequence containing the constructs has beenprepared for expression, the DNA constructs may be introduced into anappropriate host. Various techniques may be employed, such as protoplastfusion, calcium phosphate precipitation, electroporation or otherconventional techniques. After the fusion, the cells are grown in mediaand screened for appropriate activities. Expression of the sequenceresults in the production of the recombinant calpastatin proteins of thepresent invention.

E. Purification of Recombinant Calpastatin Proteins

The calpastatin proteins of this invention can be produced byfermentation of the recombinant host containing the cloned calpastatingenes. The recombinant host, such as mammalian cells producing thecloned proteins, can be grown and harvested according to techniques wellknown in the art.

The recombinant calpastatin proteins of the present invention can beextracted and purified from the recombinant host by using known proteinpurification techniques commonly employed, such as extraction,precipitation, ion exchange chromatography, affinity chromatography, gelfiltration and the like. Biochemical techniques employed to isolate thecalpastatin proteins of the present invention from neural tissue are ofparticular interest when purifying these proteins from a recombinanthost.

F. Detection of Calpastatin Proteins

The present invention also includes methods of detecting HMWC and LMWCor their functional derivatives in a sample or subject. The calpastatinproteins of the present invention may be detected with any appropriateligand, for example, an antibody which is detectably labeled.Illustrative labels include radioisotope, fluorescent, chemiluminescent,enzyme labels and the like. Methods of detecting such detectably labeledantibodies are well known to those of ordinary skill in the art and maybe performed in vitro or in vivo. For example, in vivo imaging assays asdescribed by Goldenberg et al., U.S. Pat. No. 4,444,744 (hereinincorporated by reference) may be used.

Once the calpastatins are isolated and purified, these proteins andtheir immunogenic fragments can be used as antigens to raise antibodiesspecific for the calpastatins. Methods for producing monoclonal,polyclonal, and region-specific antibodies are known to one of skill inthe art. Techniques for preparing antibodies, labeling antibodies, andperforming. immunoassays are described by Harlow et al. In: Antibodies,A Laboratory Manual (supra).

In addition, the materials for use in the assay of the invention areideally suited for preparation of a kit. Such a kit may comprise inclose confinement one or more container means such as vials, test tubes,and the like. Each of said container means comprises one of the separateelements to be used in the assay method.

For example, one of said container means may comprise antibodiesdirected against one or more calpastatin proteins. Such antibodies maybe bound to a separate solid phase immunoabsorbent or directly to theinner walls of a container. The carrier may also contain, in addition, aplurality of containers each of which comprises different, predeterminedand known amounts of antigen. These latter containers can then be usedto prepare a standard curve from which can be interpolated the resultsobtained from the sample containing the unknown amount of antigen.

Antibodies to the HMW calpastatin, described by this invention,recognized abnormalities associated with Alzheimer's disease and Down'ssyndrome. A down-regulation of CANP by an overabundance orredistribution of its inhibitors represents a reasonable and testablemechanism to account for the accumulation and/or abnormal processing ofintracellular cytoskeletal proteins.

Understanding of the endogenous inhibitors of CANP activity in humanbrain, and an ability to manipulate them, would enable the modificationor alteration of the mechanisms of atrophy and neuronal death in humanbrain, the products of neuronal degeneration, and therefore, theprocesses of pathological neurodegenerative disease, such as Alzheimer'sDisease (AD).

G. Uses of Calpastatin Proteins

Since antibodies to the calpastatins recognize abnormalities inAlzheimer's Disease (AD) brain, as well as other as yet uncharacterizedintraneuronal lesions, such antibodies would be useful in establishingthe pathological diagnosis of AD. If they recognize an early event inthe sequence of pathogenetic events leading to the death of the neuronin this and other neurofibrillary diseases, antibodies to thecalpastatin proteins of the present invention would be useful markers inthe experimental studies on mechanisms related to AD pathogenesis (i.e.,investigational tools).

Antibodies to the entire molecule or part of the molecule may be usedtherapeutically to modulate calcium-activated proteolysis in variousparts of the body. For example, calcium-activated proteolysis may beinvolved in various aspects of platelet functions, including clottingmechanisms (Noszek et al., Soc. Neurosci. Abstr. 13:1684 (1987); Moreauet al., Eur. J. Biochem. 173:185-190 (1988)) and may alter the functionof leucocytes, including their release of materials into theextracellular environment (Pontremoli et al., J. Biol. Chem.263:1915-1919 (1988)). Therefore, direct application of antibodies couldhave effects of potential clinical relevance on extracellular processes,such as blood coagulation, inflammatory responses as occur in arthritis,for example, infectious processes, and uncontrolled cell growthprocesses such as cancer. In fact, inhibitors of CANP appear to havesignificant effects in slowing the growth of certain cancers(Shoji-Kasai et al., Proc. Natl. Acad. Sci., USA 85:146-150 (1988)).

The direct use of the calpastatin molecule or a modified form withslower metabolism or greater accessibility to particular cellularcompartments (or the use of anti-calpastatin antibodies) may havepotential use in these disease states. In spinal cord trauma and otherforms of neural cell injury, CANPs are abnormally activated andsynthetic CANP inhibitors (that also inhibit other proteinases) reducethe cell damage (Siman et al., Soc. Neurosci. Abstr. 13:1684 (1987)).Since calpastatins are the only known specific inhibitors of CANP, thesepolypeptides (or derivatives of them based on their structure) may havegreater therapeutic benefit without causing cellular toxicity byinhibiting other processes. These calpastatin polypeptides may also beuseful as a basis for designing molecules that have cellular specificityin modulating CANPs.

Cell death induced by ischemia or excitations has been implicated inseveral disease states including Huntington's disease, Parkinson'sdisease, Alzheimer's Disease, etc. In each of these cases, recent dataindicate that CANPs are abnormally activated. Inhibitors of CANP such asHMWC and LMWC may therefore be useful in these conditions.

With these considerations as background, if calpastatin is structurallyor functionally abnormal in Alzheimer's Disease in a way that isimportant to the pathogenesis of AD, calpastatins have the followingpotential utility. The availability of the protein would be essential tothe development of drugs aimed at modifying the interaction ofcalpastatin with CANP (depending on the situation, enhancing orinhibiting calpastatin function). The availability of calpastatinmolecules in purified form, and antibodies thereto as described herein,enables the development of compounds modeled after the structure of thismolecule or a domain of the polypeptide. Antibodies to thesepolypeptides would be potentially useful in diagnostic applicationssince antibodies to high molecular weight calpastatin recognizepathological stuctures associated with AD. The polypeptides, or somederivative domain, could be useful in the treatment of conditions inwhich CANP activity may be abnormally increased, e.g., ischemia andcytotoxin-mediated cell death, to limit cell death due to metabolic ortraumatic insults.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified. All references described in thespecification are herein incorporated by reference in their entirety.

EXAMPLES

In the examples that follow, the materials and reagents were obtainedfrom the sources indicated.

Calpastatin was prepared from postmortem human brain provided by Dr.Edward Bird from the McLean Hospital Brain Tissue Resource Center(Belmont, Mass., U.S.A.); azocasein was obtained from Sigma Chemical Co.(St. Louis, Miss., U.S.A.); ¹⁴ C-formaldehyde (specific activity 47uCi/mmol) was purchased from New England Nuclear Corp. (Boston, Mass.,U.S.A.); diethylaminoethyl (DEAE)-cellulose (DE-52) was purchased fromWhatman BioSystems Ltd. (Maidstone, Kent, England); DEAE-Sepharose CL-4Band Sephacryl-300 was purchased from Pharmacia, Inc. (Piscataway, N.J.,U.S.A.); Ultrogel AcA-44 and Ultrogel AcA34 was purchased from IBFBiotechnics, Inc. (Savage, Md.); acrylamide and molecular weightproteins were purchased from BioRad Laboratories (Richmond, Calif.,U.S.A.); nitrocellulose membrane and horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG are products of Millipore Corp.(Bedford, Mass., U.S.A.) and Cappell Laboratories (Cochranville, Pa.,U.S.A.), respectively. Polyvinylidene difluoride (PVDF)-membrane(Immobilon-P) was purchased from Millipore Corporation (Bedford, Mass.).Peroxidase-labeled sheep anti-rabbit IgG was the product of Kirkegardand Perry Laboratories, Inc. (Gaithersburg, Md.). Other chemicals werereagent grade.

The following buffers were used in the chromatographic and assayprocedures: Buffer A: 20 mM Tris-HCl (pH 7.4), 1 mM EGTA, 2 mM EDTA, 0.5mM dithiothreitol (DTT), 1 mM benzamidine, 0.004% NAN₃, and 0.15M KCl;Buffer B: 50 mM Tris-HC1 (pH 7.4), 5 mM EDTA, 2 mM EGTA, 5 mM DTT, 2 mMbenzamidine, 0.2 mM aprotinin, and 1 mM PMSF; Buffer C: 20 mM Tris-HCl(pH 7.4), 0.15M KCl, 2 mM EDTA, 1 mM EGTA, 0.5 mM DTT, 1 mM benzamidineand 0.004% NAN₃ ; and Buffer D: 20 mM Tris-HCl (pH 7.4), 1 mM EGTA, 2 mMEDTA, 1 mM DTT, 1 mM benzamidine. CANP assays were performed in 100 mMTris (pH 7.4) and 2 mM DTT (Buffer E). The buffer used for the SDSPolyacrylamide Gel Electrophoresis was Buffer G: 25 mM Tris base, 192 mMGlycine, 0.1% SDS; and the buffer used for the transblotting apparatuswas Buffer H: 25 mM Tris base, 192 mM Glycine, 15% Methanol.

Isolation and Purification of HMW and LMW Calpastatin

Human cerebral cortex, including gray and white matter, was dissectedfrom brains of individuals without a history of neuropsychiatric diseaseand which exhibited no gross histological abnormalities. The postmorteminterval before freezing at -20° C. was 8-20 hours for all samples. Thebrains were subsequently stored for one month to 1.5 years at -70° C.before analysis.

Anion Exchange on DEAE Cellulose:

Human cerebral cortical tissue (200-1200 g) was homogenized in fourvolumes of cold buffer B. All subsequent steps were performed at 0°-4°C. After centrifugation at 16,000×g, the protein was concentrated byprecipitation in 35-65% ammonium sulfate. The precipitate was collectedby centrifugation and resuspended in 4 volumes of buffer B with gentleshaking. This solution was dialyzed against buffer B for 48 hours andthen applied to a 4.4×30 cm column of DEAE-cellulose (DE-52)equilibrated with buffer B. After extensive washing, the column waseluted with a 4000 ml KCl gradient (0-0.4M) in buffer B at a flow rateof 100 ml/hr.; 15 ml fractions were collected. Fractions containinginhibitor activity were pooled and the protein was concentrated byammonium sulfate precipitation between 35-65% ammonium sulfate asdescribed above.

Gel Chromatography on Ultrogel AcA44:

The calpastatin fractions in the previous stage (DEAE-cellulose) wereapplied to a column (4.4×90 cm) of Ultrogel AcA44 equilibrated withbuffer C. The column was eluted with the above buffer at a flow-rate of35 ml/hr and 13 ml fractions were collected in each tube. Two CANPinhibitory activities were eluted from the Ultrogel AcA44 column. Theinhibitor fractions having an inhibitory activity greater than 50% werecombined as Fraction I (HMWC) and Fraction II (LMWC), from earlier andlater inhibitory peaks, respectively. Fraction I (HMWC) was dialyzedagainst buffer D at 0° C.

DEAE-Sepharose CL 4B Chromatography:

The dialyzate (HMWC) from the previous step (Ultrogel AcA44) was appliedto a column (2.5×25 cm) of DEAE-Sepharose CL 4B equilibrated with bufferD. After the column was washed with 800 ml of buffer D, the column waseluted with a linear gradient of 0-0.4M KCl (total 700 ml) in buffer D.The flow rate was 50 ml/hr, and 10 ml fractions were collected. Theinhibitor fractions eluted with KCl gradient were pooled andconcentrated with the aid of Centriprep-10 (Amicon Corp.).

Ions-exchange chromatography with CM cellulose:

The pooled fractions of LMWC obtained from Ultrogel AcA-44 column werepurified over a CM-cellulose ion-exchange column at pH 5.0. Pooled LMWCwas dialyzed against 20 mM Na-acetate, pH 5.0, 1 mM GGTA, 2 mM EDTA, 2mM benzamidine at 0° C. The dialyzate was applied to a column (2.5×25cm) of CM-cellulose (CM-52, Whitman) equilibrated with buffer E. Afterthe column was washed with 800 ml of buffer E, the column was elutedwith a linear gradient of 0-0.4M NaCl (total 700 ml) in buffer E. Theflow rate was 50 ml/hr, and 10 ml fractions were collected. Theinhibitor fractions were pooled and concentrated with the aid ofCentriprep-10 (Amicon Corp.).

Affinity Chromatography of LMWC on Con A-Sepharose:

The inhibitor fractions from CM-cellulose were pooled and concentratedfor the next purification step. The concentrated sample was loaded to acolumn (1.5×5 cm) of Con A-Sepharose equilibrated with 20 mM Tris-HCl,pH 7.4, 1 mM benzamidine and 0.5M NaCl. After the column was washedextensively with the buffer, the column was eluted with 0.5M methylα-D-glucopyranoside in 20 mM Tris-HCl, pH 7.4 and 1 mM benzamidine. TheLMWC was recovered in the non-binding fraction whereas α PI wasseparated from LMWC into the binding fraction.

Heat treatment:

The HMWC inhibitor obtained from the DEAE-Sepharose CL-4B column washeated at 100° C. for 5 min, cooled to 0° C., and centrifuged at100,000×g for 30 min at 4° C.

The LMWC inhibitor obtained from the Con A-Sepharose column was heattreated as above.

Gel chromatography:

After heat treatment, the HMWC supernatant from the heat treatment stepwas applied to a column (2.5×115 cm) of Ultrogel AcA34 equilibrated withbuffer C. A flow rate of 23.0 ml/hr was used and 5 ml fractions werecollected in each tube. In contrast, the heat treated supernatantcontaining LMWC was applied to a Sephacryl S-300 column (2.5×120 cm)equilibrated with buffer C. The column was run with a flow rate of 12.5ml/hr and 5 ml fractions were collected.

II. Assay of Protein Concentration and Inhibitor Activity

[¹⁴ C]-azocasein was prepared by reductive alkylation with [¹⁴C]-formaldehyde (Dottavio-Martin et al., Anal. Biochem. 87:562-565(1978)). Calpastatin activity was expressed in terms of ability todecrease the activity of human brain CANP. Millimolar calcium-dependentproteinase was purified to homogeneity from postmortem human brain aspreviously described (Vitto et al. J. Neurochem. 47:1039-1051 (1986).Fractions containing inhibitor activity were pre-incubated with mCANP.After a 10-min preincubation at 4° C. the reaction was initiated by theaddition of calcium chloride in a reaction mixture containing 50 mMTris-HCl (pH 7.4), 5 mM CaCl₂, 1 mM DTT, 0.25% (W/V) azocasein plus [¹⁴C]-azocasein (specific activity: 133 cpm/ug [¹⁴ C-azocasein]). Afterincubation for 30 min at 30° C., the reaction was terminated with 300 μLof cold 10% trichloroacetic acid. Enzyme activity was measured as theradioactivity in the acid-soluble fraction after centrifugation aspreviously described (Nixon, Brain Res. 200:69-83 (1980)). Acid-solubleradioactivity was determined in samples lacking enzyme, and this valuewas subtracted from the experimental samples as non-enzymatic"background." In the absence of inhibitor, the rate of azocaseinhydrolysis was constant during 30 min of incubation and increased inproportion to the amount of enzyme protein. Specific calcium-dependentproteinase inhibitor activity was calculated from curves describing theinhibition of CANP activity with increasing amounts of inhibitorprotein. One unit of inhibitor activity was defined as the amount ofprotein required to inhibit 50% of the activity of 0.5 mg of purifiedCANP. In cases where the presence of inhibitor but not its specificactivity was required, inhibitory activity was measuring using a humanbrain mCANP fraction partially purified by DE-52 chromatography (Vittoet al. J. Neurochem. 47:1039-1051 (1986)). The amount of proteinaseinhibitor added to the assay was adjusted to yield approximately 50%inhibition of CANP.

III. Additional Methods

Estimation of Molecular Weight of Calpastatin on Ultrogel AcA34

The native molecular weight of calpastatin was estimated on a column(2.5×115 cm) of Ultrogel AcA34. The column was pre-equilibrated andeluted with buffer C at a flow rate of 23 ml/hr. Fractions of 5 ml werecollected in each tube. The following protein standards were used todetermine the molecular weight of calpastatin proteins: Catalase (M_(r)=240,000), aldolase (M_(r) =158,000), bovine serum albumin (M_(r)=68,000), ovalbumin (M_(r) =45,000), chymotrypsinogen A (M_(r) =25,000),and ferritin (450,000).

Estimation of Molecular Weight of Calpastatin on Sephacryl S-300

The native molecular weight of calpastatin (LMWC) was examined on acolumn (2.5×120 cm) of Sephacryl S-300. The column was pre-equilibratedand eluted with buffer C at a flow rate of 15 ml/hr. Fractions of 5 mlwere collected in each tube. The molecular weight was determined byusing following standards, Aldolase (M_(r) =158,000), bovine serumalbumin (68,000), ovalbumin (45,000), and chymotrypsinogen A (M_(r)=25,000).

Polyacrylamide Gel Electrophoresis

Samples were analyzed by electrophoresis in sodiumdodecylsulfate-polyacrylamide (SDS-PAGE) slab gels by the method ofLaemmli (Nature 227:680-685 (1970)). The standard proteins,phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase,soybean trypsin inhibitor, and lysozyme of molecular weights 92,500,68,000, 45,000, 31,000, 21,500, and 14,400, respectively, were used.

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis was performed according to O'Farrell(J. Biol. Chem. 250:4007-4027 (1975)). Gels were stained for proteinswith Coomassie Blue, or proteins from gels were electrophoreticallytransferred to PVDF-membranes or nitrocellulose membranes andimmunostained with affinity purified antibody (I-2-7) to the highmolecular weight calpastatin.

Isoelectric Focusing of Purified Calpastatins

Purified calpastatins were subjected to isoelectric focusing in 6.5%polyacrylamide gel containing 2.5% ampholine (pH 3-8), 1.6M urea, and0.5% NP-40 at 4° C. The gels were sliced into 3 mm segments, and eachsegment was assayed for calpastatin by the standard assay protocol.

Amino Acid Analysis and Protein Sequencing

High molecular weight and low molecular weight calpastatins werepurified as demonstrated in FIGS. 1 and 2. Aliquots of each purifiedfraction were subjected to SDS-polyacrylamide gel electrophoresis (10%polyacrylamide gel containing 0.1% SDS in Laemmli's system (Nature227:680-685 (1970)). Proteins on the gels were electrophoreticallytransferred to PVDF (polyvinylidine difluoride)-membrane (Immobilon-P)in 10% methanol CAPS (3-(cyclohexylamino)-1-propanesulfonic acid) buffer(pH 11.0) system. The proteins were identified on the membrane bystaining with Coomassie Brilliant Blue R. The appropriate bands wereexcised from the membranes and subjected to amino acid analysis andsequence analysis (Matsudaira, J. Biol. Chem. 262:10035-10038 (1987)).Automated Edman degradation was performed on an Applied Biosystems Model470-AA gas liquid-phase protein sequencer. PTH-amino acids recovered ateach cycle of the Edman degradation were analyzed equipped on-line withan Applied Biosystems Model 120-A HPLC.

Immunoblotting

Immunoblotting was performed according to Towbin et al. (Proc. Natl.Acad. Sci. USA 76:4350-4354, 1979) in transfer buffer containing 15%MeOH, 25 mM Tris, and 192 mM glycine (pH 8.3) by applying constantcurrent (0.5 A) at 2° C. Proteins were electrophoretically transferredonto PVDF-membrane or nitrocellulose-membrane. Non-specific backgroundwas removed by incubating the membrane with 20 mM Tris-HCl (pH 7.4),plus 0.5M NaCl and 5% non-fat dried milk blocking solution for 1 hr atroom temperature. Purified sheep IgG (1 mg/ml) to human brain HMWCcalpastatin (I-2-7) was diluted 1:10-1:1000 in blocking buffer andincubated with the transfer membrane overnight at 4° C.Peroxidase-conjugated rabbit anti-sheep IgG was diluted 1:1000 (stocksol. of 1 mg IgG/ml) in blocking solution and membranes were incubatedfor 5 hr at room temperature. After several washes of the membranes withTBS, peroxidase activity was visualized with H₂ O₂ and 0.001%4-chloro-1-naphthol plus N,N'-dimethyl-p-phenylenediamine according tothe method developed by Kobayashi and Tashima (Anal. Biochem. 183:9-12(1989)).

Immunocytochemistry

Postmortem human brains from individuals with Alzheimer's disease (AD)and age-matched (62 to 78 years) neurologically normal brains were usedin this study. CNS tissue was procured from the McLean Hospital BrainTissue Resource Center (Belmont, Mass.) and Massachusetts GeneralHospital (Boston, Mass.). Premortem clinical diagnosis of Alzheimer'sdisease was confirmed neuropathologically. Control brains were obtainedfrom individuals with no history of neuropsychiatric disease andexhibited no gross or microscopic histopathology. The postmorteminterval before immersion fixation in 10% phosphate-buffered formalinfor all specimens used in this study was 12 to 24 hours.

Tissue blocks (3×1×0.4 cm) from the prefrontal cortex of fixed brainswere cut into 30 to 35 μ-thick sections using a Lancer Series 1000vibratome. Brain sections were treated with Nissl and/or Bielshowskystains for routine histological inspection or were processed forimmunocytochemistry. Additional vibratome sections were immunostainedusing a rhodamine-conjugated goat-anti rabbit secondary antibody and/orstained with thioflavin S (Cappel/Worthington Biochemicals, CooperBiomedials, Malvern, Pa.) to verify the presence or absence of pairedhelical filaments or senile plaques.

For histological demonstration of anti-calpastatin immunoreactivities, amodification of the avidin-biotin complex (ABC) method of Hsu et al. (J.Histochem. Cytochem. 29:577-580 (1981)) was employed as follows: 1)free-floating sections were incubated for 30 minutes at room temperaturein CH₃ OH with 0.3% H₂ O₂ ; 2) sections were washed 3 times for 10minutes each in "diluting solution" which consisted of 20 mMTris-buffered saline (TBS)-pH 7.4, 0.4% Triton X-100, 2% bovine serumalbumin (BSA), 1% normal goat or rabbit serum, and 0.9% NaCl; 3) tissuesections were blocked for 30 minutes at room temperature with 20% normalgoat or rabbit serum in 20 mM TBS; after blocking, sections wereincubated from 12-72 hours at 4° C. in anti-calpastatin antisera usingdilutions of 1:250-1:1000. Serial dilutions of antisera were made in"diluting fluid"; 5) sections were washed 3 times for 10 minutes each indiluting fluid; 6) following incubation in primary antisera, sectionswere incubated for 30 minutes at room temperature in 20 mMTBS-biotinylated secondary antibody (either biotinylated goatanti-rabbit or rabbit anti-sheep), 45 microliters/10 cc) in 20 mM TBS);7) sections were washed 3 times for 10 minutes each in diluting fluidand subsequently incubated for 60 minutes at room temperature in avidin(90 microliters/10 cc)-biotin (90 microliters/10 cc) in 20 mM TBS);sections were washed 3 times in diluting solution. All biotinylatedsecondary antibodies and avidin-biotin reagents were obtained fromVector Laboratories, Inc., Burlingame, Calif. Following washes, tissuesections were incubated for 5 minutes in diaminobenzidine (1 mg/ml) in10 mM TBS-0.02% H₂ O₂ at room temperature; sections were washed 3-5minutes in distilled water and rinsed 1-3 minutes in 20 mM TBS.

IV. Results of Purification and Characterization

Purification of HMWC and LMWC Calpastatin

Table 1 summarizes the purification data of HMW human brain calpastatin(41 Kda calpastatin). More than 19% of the inhibitory activity wasrecovered and the 41 Kda calpastatin was purified 964-fold over theDEAE-cellulose partially purified inhibitor. The final specific activityof purified material was approximately 36,000 units/mg protein. As seenin Table 1, heat-treatment and Ultrogel AcA34 steps were very effectivefor the purification and increased the purification in each step morethan 24-fold and 22-fold, respectively.

Although some calpastatin activity is detectable in the crudehomogenate, a fair amount of endogenous calpain activity disturbs anaccurate determination of inhibitory activity. We therefore introducedion-exchange chromatography on DEAE-cellulose (DE-52) to separatecalpain and calpastatin. The column was eluted with a linear saltgradient (FIG. 3). Inhibitory activity was eluted at a saltconcentration between 0.05 and 0.15M KCl from the column as a singlepeak of activity. The mCANP was eluted at 0.25-0.3M KCl in a separatedfraction from calpastatin.

The pooled calpastatin fraction after DEAE-cellulose chromatography wasapplied to further purification on Ultrogel AcA44 column chromatography.FIG. 4 shows a typical elution profile of DEAE-cellulose fraction onUltrogel AcA44 and demonstrates that calpastatin(s) is eluted as twopeaks of inhibitory activity (fraction U-1, and U-2. The earliesteluting peak (U-1) corresponding to HMWC contained approximately 60% ofthe total activity. The second peak of activity (U-2) corresponding toLMWC contained about 40% of the total inhibitory activity. Purificationand characterization of these fractions (U1 and U2) is discussed below.

                                      TABLE 1                                     __________________________________________________________________________    Purification of High Molecular Weight Calpastatin from Human Brain                              Total                                                                         Activity                                                                           Total   Specific                                                    Volume                                                                             (Units)*                                                                           Protein                                                                           Yield                                                                             Activity                                                                            Purification                             Purification Stage                                                                         (ml) × 10-3                                                                       (mg)                                                                              (%) (units/mg)                                                                          (-fold)                                  __________________________________________________________________________    Human Brain Homogenate                                                                     3600                                                             DEAE-cellulose Pooled                                                                      1080 55.0 1500                                                                              100  37   1                                        Fraction (DE-I)                                                               Ultrogel AcA 44                                                                             230 25.3 550 46   46   1.2                                      Fraction I (U-1)                                                              DEAE-Sepharose CL4B                                                                         80  18.6 282 34   66   1.8                                      Heat-treatment (100° C., 5                                                            8  18.0 11.3                                                                              33  1593  43.1                                     min) 100,000 × g                                                        Supernatant                                                                   Ultrogel AcA 34                                                                             15  10.7 0.3 19.5                                                                              35667 964                                      __________________________________________________________________________     *One unit of inhibitor activity was defined as the inhibition of 50% of       the activity of 0.5 μg of CANP.                                       

a. Purification of HMWC

The active fractions of U-1 were pooled, dialyzed and applied to thesecond ion-exchange chromatography on DEAE-Sepharose CL 4B which waseluted with a linear gradient of KCl. Inhibitory activity elutes withthat position of the gradient at a salt concentration between 0.15 and0.23 from this column.

The pooled inhibitor fraction after DEAE-Sepharose CL-4B chromatographywas heat-treated at 100° C. for 5 min. The boiled fraction wascentrifuged at 100,000×g for 30 minutes and the supernatant wasrecovered as inhibitor fraction. Interestingly, more than 95% ofcontaminated protein was removed by this treatment, and 100% ofinhibitory activity was recovered in the supernatant. The heatedfraction was further analyzed on Ultrogel AcA34 column chromatography.The inhibitory activity was recovered as a single symmetrical peak fromthis column at the position of 300 kDa protein (FIG. 6). The molecularsize of 300 kDa did not change with or without heat-treatment of thisfraction on Ultrogel AcA34 under a non-denaturing condition.

b. Purification of LMWC

The U-2 active fraction corresponding to LMWC was subjected toion-exchange chromatography on CM cellulose (CM-52) (FIG. 11). Theinhibitor assay and protein determination profiles of the elutedfractions from CM-cellulose demonstrated that most of contaminatingproteins in low molecular weight calpastatin, however, did not bind tothis matrix (FIG. 11). The pooled fraction was further purified over aCon A-Sepharose column. This chromatographic step was followed byheat-treatment and final gel-chromatography on Sephacryl S-300 resultedin the complete purification of the LMWC calpastatin. LMWC was purifiedas a single peak of activity at the position of 60 kDa protein onSephacryl S-300 under non-denaturing condition (FIG. 2). Table 2 showsthe results of purification of LMWC. The LMWC was purified 527-fold overthe Ultrogel AcA44 partially purified LMWC, and the final specificactivity was 19500 u/mg. FIG. 2 displays a flow diagram of thepurification of low molecular weight calpastatin from human brain.

Preliminary studies to investigate the molecular size of native lowmolecular weight calpastatin demonstrated that inhibitor appears to havea broad molecular distribution in its molecular weight of 40-80 kDa onUltrogel AcA34 and 40-100 kDa on Sephadex G-200 under non-denaturingcondition. This wide range of distribution of inhibitor activitysuggested that low molecular weight calpastatin might be highlyglycosylated as a result of posttranslational modification of thisprotein in vivo.

                                      TABLE 2                                     __________________________________________________________________________    Purification of Low Molecular Weight Calpastatin from Human Brain                               Total                                                                         Activity                                                                           Total   Specific                                                    Volume                                                                             (Units)*                                                                           Protein                                                                           Yield                                                                             Activity                                                                            Purification                             Purification Stage                                                                         (ml) × 10-3                                                                       (mg)                                                                              (%) (units/mg)                                                                          (-fold)                                  __________________________________________________________________________    Human Brain Homogenate                                                                     3600                                                             DEAE-cellulose                                                                             1080 55.0 1500                                                                              100  37   1                                        Pooled Fraction (DE-I)                                                        Ultrogel AcA 44 Fraction                                                                    210 20.1 315 37   63   1.7                                      II (U-2)                                                                      CM-cellulose  45  15.2 16.8                                                                              28   905  24.5                                     Heat-treatment (100° C.,                                                              5  13.4 9.2 24  1457  39.4                                     10 min) 100,000 × g                                                     Supernatant                                                                   Sephacryl S-300                                                                             25  8.2  0.42                                                                              15  19523 527.6                                    __________________________________________________________________________     *One unit of inhibitor activity was defined as the inhibition of 50% of       the activity of 0.5 μg of CANP.                                       

Immunoblotting and Isoelectric Focusing of Purified HMW and LMWCalpastatins

Purified HMWC was further examined by 2D-gel protein analysis followedby immunoblotting. When inhibitory activity was examined with mCANP/[¹⁴C]-azocasein system in the extracts from isoelectric focusedpolyacrylamide gels (1 dimensional analysis), a single peak ofinhibitory activity was demonstrated at the position of pI=4.5 (FIG. 8).Affinity-purified polyclonal antibody I-2-7 identified a single peptidehaving very acidic pI (pI=4.5) at 41 Kda by immunoblotting (FIG. 7).

The protein staining by silver-staining on SDS-PAGE after thetwo-dimensional process demonstrated a single spot which corresponded to41 Kda immunoreactive protein on the same gels.

As seen in FIG. 13, purified low molecular weight calpastatin appearedas two peaks on isoelectric focusing gels. The isoelectric point of LMWCwas in the range of 4.2-4.7.

Heat-stability

Since calpastatins from other sources exhibit remarkable heat stability,we examined the activity of high molecular weight and low molecularweight brain calpastatin after a 5-min exposure to a temperature of 100°C. As seen in Table 1 and 2, even after treatment at 100° C. for 5 min,the inhibitory activity was stable.

Low concentrations of purified LMW inhibitor were capable of inhibitingpurified human brain mCANP. Both forms of purified human braincalpastatin (HMWC and LMWC) inhibited purified mouse brain mCANP and amicromolar calcium-dependent CANP activity in mouse retinal ganglioncell axons (Nixon et al., J. Neurosci. 6:1252-1263 (1986)). Neitherinhibitor had effects on the activities of trypsin, chymotrypsin,bromolain, papain, and purified human brain cathepsin D, when assayed asdescribed above.

Stoichiometry of mCANP and Calpastatin Interaction

The availability of both purified mCANP and purified calpastatins madeit possible to study the number of proteinase molecules inactivated byone calpastatin polypeptide simply by mixing a known ratio of mCANP andcalpastatin and assaying for remaining proteinase activity. Thedose-dependence of the inhibitor of mCANP by HMWC is shown in FIG. 9A.The values of ID₅₀ (the dose of calpastatin that caused 50% inhibition)were calculated to be 0.13×10⁻³ mM on 2×10⁻³ mM mCANP. One molecule of300 Kda (HMWC) calpastatin, therefore, could bind approximately 16calpain molecules.

In contrast, the ID₅₀ value of LMWC was 0.3×10⁻³ mM on 2×10⁻³ mM mCANP(FIG. 9B). The binding ratio of LMWC to mCANP is estimated to be 6calpain molecules.

Thus, 300 kDa calpastatin has a calpain binding capacity 2.6-2.7 timesthat of 60 kDa calpastatin.

Amino Acid Analysis and Sequence Analysis of Calpastatin Proteins fromHuman Brain

HMWC demonstrates a relatively high content of acidic residues, such asaspartic acid and glutamic acid, and neutral aliphatics such as glycine,alanine and serine (Table 3). The amino acid composition of HMWC wascompared with the two different calpastatins, pig heart calpastatin (107kDa protein) and rabbit liver calpastatin (68 kDa protein) (Table 4).NH₂ -terminal sequence analysis was performed in triplicate on 25 pmoleof HMWC through 19 cycles. The sequence as shown in Table 5 is asfollows:

NH₂-X-Met-Pro-Pro-Glu-Pro-Ala-Thr-Leu-Lys-Gly-X-Val-Pro-Asp-Asp-Ala-Val-Glu(SEQ ID NO: 1)

A computer search using a protein data base (program Univ. Wisconsingenetic groups) revealed that a part of the amino acid sequence of HMWCshowed some homology to other calpastatins, such, as pig heartcalpastatins and rabbit liver calpastatins. Although the size of thesethree proteins varies somewhat, the homology of the corresponding aminoacid sequences between pig heart calpastatin and human brain HMWC,rabbit liver calpastatin and human brain HMWC was 36% and 34%,respectively.

Table 6 shows the amino acid composition of LMWC. Amino-terminalsequence analysis of LMWC is shown in Table 7.

An additional comparison of acid compositions was made between brainHMWC and human liver calpastatin as deduced from the complete genesequence (Table 4A). Since the N-terminal 19 amino acids of brain HMWCcorresponds precisely to an internal sequence in the cDNA of livercalpastatin, we compared the amino acid composition of brain HMWC tothat of the C-terminal end of human liver calpastatin (approximately a41-kDa polypeptide) starting at the beginning of the homologous internalsequence (Table 4A). The clear differences between this fragment ofliver calpastatin and the 41-kDa HMWC indicate that the braincalpastatin is distinct from the liver calpastatin, despite regions ofmarked homology.

                  TABLE 3                                                         ______________________________________                                        Amino Acid Analysis of High Molecular                                         Weight Calpastatin.sup.1                                                                       HMWC (41 kDa).sup.2                                          Amino Acid       mole (%)                                                     ______________________________________                                        Asx.sup.3    (D,N)   15.1                                                     Thr          (T)     2.5                                                      Ser          (S)     10                                                       Glx.sup.4    (E,Q)   16.7                                                     Pro          (P)     4.7                                                      Gly          (G)     11.1                                                     Ala          (A)     9                                                        Cys/2        (C)     0                                                        Val          (V)     3.2                                                      Met          (M)     1.8                                                      Ile          (I)     0                                                        Leu          (L)     7                                                        Tyr          (Y)     1.5                                                      Phe          (F)     1.9                                                      Lys          (K)     10.5                                                     His          (H)     1.6                                                      Arg          (R)     3.3                                                      Try          (W)     --                                                       ______________________________________                                         .sup.1) HMWC was purified on an Ultrogel AcA34 column as 300 kDa protein      under nondenaturing conditions. Purified HMWC was subjected to SDSPAGE an     protein was transferred to the PVDFmembrane from the gel. Amino acid          composition was directly determined from the membrane according to            Matsudaira (J. Biol. Chem. 262, 10035, 1987).                                 .sup.2) Molecular weight of HMWC was estimated on SDSPAGE under reduced       conditions.                                                                   .sup.3) Asx = sum of aspartic acid (D) and asparagine (N).                    .sup.4) Glx = sum of glutamic acid (E) and glutamine (Q).                

                  TABLE 4                                                         ______________________________________                                        Comparison of the amino acid                                                  composition of human brain HMWC and                                           other Ca.sup.2+ activated neutral proteinase inhibitors                                                Pig       Rabbit                                                              heart     liver                                                  HMWC (41 kDa)                                                                              calpastatin.sup.1                                                                       calpastatin.sup.2                          Amino Acid  mole (%)     mole %    mole %                                     ______________________________________                                        Asx    (D,N)    15.1         10.3    10.3                                     Thr    (T)      2.5          6.4     4.8                                      Ser    (S)      10           9.0     8.7                                      Glx    (E,Q)    16.7         14.3    15.7                                     Pro    (P)      4.7          11.1    10.2                                     Gly    (G)      11.1         6.7     6.5                                      Ala    (A)      9            7.6     12.7                                     Cys/2  (C)      0            0.5     0                                        Val    (V)      3.2          5.2     3.3                                      Met    (M)      1.8          0.8     1.4                                      Ile    (I)      0            2.7     7.7                                      Leu    (L)      7            7.5     0.9                                      Tyr    (Y)      1.5          0.7     1.1                                      Phe    (F)      1.9          1.4     11.3                                     Lys    (K)      10.5         10.8    0.8                                      His    (H)      1.6          1.0     3.0                                      Arg    (R)      3.3          4.0     --                                       Try    (W)      --           0                                                ______________________________________                                         .sup.1) Amino acid composition was taken from Murachi's group (Biochem. J     235, 97, 1986).                                                               .sup.2) Amino acid composition was taken from Suzuki's group (Proc. Natl.     Acad. Sci. 84, 3590, 1987).                                              

                  TABLE 4A                                                        ______________________________________                                        Human Liver Calpastatin:                                                      deduced AA compositions                                                                      41 kDa    42 kDa   Human                                       Amino Total    C-terminal                                                                              N-terminal                                                                             Brain  Amino                                Acid  Sequence Fragment  Fragment HMWC   Acid                                 ______________________________________                                        A ala 6.6      6.3       6.8      9      A ala                                V val 3.5      2.9       4        3.2    V val                                L leu 7        7.9       6.4      7      L leu                                I ilu 2        1.4       2.5      0      I ilu                                G gly 3        2.9       3.2      11.1   G gly                                W trp 0        0         0        0      W trp                                Y tyr 1.3      1         1.5      1.5    Y tyr                                F phe 1        1.3       0.7      1.9    F phe                                H his 0.9      0.8       1        1.6    H his                                S ser 8.5      7.2       9.6      10     S ser                                T thr 5.3      5.5       5.1      2.5    T thr                                P pro 8.8      9.1       8.6      4.7    P pro                                C cys 0.9      0.7       1        0      C Cys                                M met 1.4      1.2       1.6      1.8    M met                                R arg 3.7      3.3       4.1      3.3    R arg                                K lys 16.4     16.7      16.1     10.5   K lys                                E glu 14.2     11.2      16.6     16.7   E glu                                Q gln 3.7      4         3.4      --     Q gln                                D asp 10.5     14.9      7.1      15.1   D asp                                N asn 1.3      1.8       0.8      --     N asn                                ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Amino-terminal Sequence of High Molecular Weight Calpastatin                  of Human Brain                                                                ______________________________________                                        1    2      3      4    5    6    7    8    9    10                           X    Met    Pro    Pro  Glu  Pro  Ala  Thr  Leu  Lys                               M      P      P    E    P    A    T    L    K                            11   12     13     14   15   16   17   18   19                                Gly  X      Val    Pro  Asp  Asp  Ala  Val  Glu (SEQ ID                                                                   NO: 1)                            G    X      V      P    D    D    A    V    E                                 ______________________________________                                         Residues are numbered from the amino terminus.                           

                  TABLE 6                                                         ______________________________________                                        Amino Acid Analysis of Low Molecular Weight Calpastatin.sup.1                                   LMWC                                                                          KDa peptide                                                 Amino Acid        mole %                                                      ______________________________________                                        Asx.sup.3     (D,N)   8.4                                                     Thr           (T)     3.6                                                     Ser           (S)     10.3                                                    Glx4          (E,Q)   17.1                                                    Pro           (P)     5                                                       Gly           (G)     16.4                                                    Ala           (A)     6.4                                                     Cys/2         (C)     0                                                       Val           (V)     4.3                                                     Met           (M)     0.9                                                     Ile           (I)     3.3                                                     Leu           (L)     3.1                                                     Tyr           (Y)     2.7                                                     Phe           (F)     7                                                       Lys           (K)     1.3                                                     His           (H)     4                                                       Arg           (R)     --                                                      Try           (W)                                                             ______________________________________                                         .sup.1) LMWC was purified on a Sephacryl S300 column under nondenaturing      conditions. The LMWC peptide was identified on SDSPAGE. The peptide was       transferred to the PVDFmembrane from the gels. Amino acid composition of      the peptide on the PVDFmembrance was determined by the methods of             Matsudaira (J. Biol. Chem. 262, 10035, 1987).                                 .sup.2) Expressed as residues/100 residues.                                   .sup.3) Asx = sum of aspartic acid (D) and asparagine (N).                    .sup.4) Glx = sum of glutamic acid (E) and glutamine (Q).                

                  TABLE 7                                                         ______________________________________                                        N-Terminal Amino Acid Sequence of Low Molecular                               Weight Calpastatins of Human Brain                                            ______________________________________                                         ##STR1##                                                                      ##STR2##                                                                     (SEQ ID NO: 2)                                                                ______________________________________                                    

V. Antibodies to HMW and LMW Calpastatin

Antibody Production

Pure samples of the LMW inhibitor are purified on a two-dimensional SDSpolyacrylamide gel and are excised from the gel. The slices areequilibrated to pH 6.8 in 125 mM Tris buffer containing 0.1% SDS, aremixed with an equal amount of complete Freund's adjuvant, and areinjected subcutaneously and intradermally into a female rabbit asdescribed by Vitto et al., J. Neurochem. 47:1039-1051, 1986. Injectionsof 50 mg of protein in 2 ml of emulsion are given in monthly intervals,using complete Freund's adjuvant initially, and incomplete Freund'sadjuvant in subsequent injections. Animals are bled 7-10 days afterinjection. The blood is allowed to clot and is spun to obtain serum(Vitto et al., J. Neurochem. 47:1039-1051, 1986). Additional antibodiesare prepared by immunizing sheep with gel slices containing the purifiedpolypeptide using the same procedure described above for rabbit exceptthat 50-100 mg of protein are used for each immunization.

Antibodies to HMWC were prepared in sheep by injecting the purified41-kDa polypeptide electrophoretically separated on 5-15%SDS-polyacrylamide gels. Antibodies against HMWC were generated andcharacterized. I-2 is a sheep anti-serum raised against a partiallypurified 41-kDa polypeptide of HMWC. Antibody specific for the purified41-kDa HMWC was prepared by antigen affinity chromatography. Purifiedpreparations of HMWC were electrophoretically separated andelectrotransferred to PVDF membranes. Regions of the membrane containingthe 41-kDa HMWC were cut into strips (antigen strips). Antigen stripswere incubated for 1 hour at room temperature with 5% non-fat dry milksolution containing 2 mM EGTA, 0.15M NaCl in 50 mM Tris-HCl pH 7.4(blocking buffer). Strips were incubated with I-2 antiserum for 1 hourat room temperature and then washed 3 times in TBS at room temperature(RT) for 5 minutes. Bound antibody was eluted from the antigen strips byincubation with 1.5 ml 0.1M glycine-HCl pH 3.0 for 10 minutes at roomtemperature which was immediately neutralized with 40 μL of of 1M TrispH 11.0. Antibody solutions were concentrated using Centriprep 10filtration units (Amicon Corp.) and stored at -70° C.

In some experiments, a second affinity purified antibody was preparedfrom I-2 using as the ligand the 87-kDa protein band present on gels ofpurified HMWC in low levels relative to 41-kDa HMWC. I-2 affinitypurified antibodies to either the 41-kDa or 87-kDa protein recognizedboth the 41-kDa and the 87-kDa proteins although the 87-kDa antibodyyielded a somewhat stronger signal with the 87-kDa antigen compared tothe 41-kDa antigen. Studies detailed below involve the use of theaffinity-purified I-2 antibodies (designated I-2 (41) or I-2 (87))unless otherwise indicated.

Immunoblot Analysis

Immunoblot analysis using I-2 affinity purified antiserum demonstratedstrong immunostaining of the protein band corresponding to HMWCthroughout its purification. Immunoreactivity in different fractions ofeach chromatography step correlated well with the relative inhibitoryactivity of these fractions. Partially purified fractions of LMWCreacted strongly with rabbit antiserum to LMWC (I-1) but were notimmunostained by the antiserum to HMWC (I-2). Conversely, a partiallypurified fraction of HMWC reacted with I-2 but not I-1. These resultsindicate the unrelatedness of HMWC and LMWC by immunologic criteria,confirming biochemical data described above.

Partially purified fractions of calpastatin were prepared fromsupernatant extracts of various neural and non-neural tissues by ionexchange chromatography on DEAE cellulose (step 1 of the calpastatinpurification scheme). The fractions containing CANP inhibitory activitywere eluted at 0.15M KCl and tested by immunoblot analysis. In somecases, the preparations were further enriched by heat treatment at 100°C. for 10 minutes to remove heat unstable proteins, which precipitatedand were eliminated by centrifugation. The I-2 (41) antisera recognizedstrongly cross-reactive bands in the inhibitory fractions which variedin molecular weight for each tissue.

In brain samples from rabbit, monkey and human, I-2 (41) recognized a41-kDa protein as a major immunoreactive constituent. In addition, I-2(41) also strongly labeled a 68-kDa protein in human erythrocytescorresponding to the molecular mass of the known calpastatin in thistissue and a 50- to 55-kDa protein of unknown relation to calpastatin.In rat heart and liver, a 100- to 105-kDa immunoreactive protein wasobserved, which may correspond to the larger calpastatin formspreviously reported in some non-neural tissue including liver. Theseresults demonstrate the widespread occurrence of proteinsimmunologically related to the brain HMWC in non-neural tissues. Theyalso demonstrate that a 41-kDa calpastatin is the major immunoreactiveform of HMWC in brains from various mammalian species. This indicatesthat the 41-kDa form of brain HMWC is not an artifact of postmortemprocessing since rabbit and monkey brain was snap frozen within 20minutes of death, comparable to the post-mortem interval for thenon-neural tissue samples.

It should be noted that in highly purified fractions of human HMWC, I-2(41) and especially I-2 (87) also recognize three minor protein bands at87, 65 and 55 kDa. Only the 87-kDa form (and 41-kDa form) can bedetected on Coomassie stained gels. We suspect that these forms may beproforms of the 41-kDa HMWC.

VI. Postmortem Stability of Calpastatins in Bovine Brain

The presence and content of polypeptides immunoreactive againstantibodies to LMWC and HMWC was measured in samples of adult bovinecerebral cortex. Changes in the content of LMWC and HMWC in bovine brainwere analyzed in samples of cerebral cortex dissected from calf brainremoved with 10 minutes after death and maintained for various intervalsat room temperature.

The results demonstrate the remarkable stability of HMWC and LMWC inpostmortem tissue. During a 30-hour postmortem interval at roomtemperature, there was less than 10% reduction in the content of the41-kDa component of HMWC and the 26-30 kDa components of LMWC.

VII. Immunocytochemical Studies with Affinity Purified Antisera RaisedAgainst Calpastatin Polypeptide: Detection of Abnormalities inAlzheimer's Disease Brain

Immunocytochemical findings using affinity purified antibodies to HMWCprovide evidence supporting the involvement of CANP systems in ADpathogenesis. I-2 (41) and 1-2 (87) yielded identical immunocytochemicalstaining patterns in all brain regions of Alzheimer and control tissueas well as in mouse brain and monkey. In vibratome sections of neocortexfrom each of these species, immunoreactivity to HMWC was predominantlylocalized in neuronal perikarya and apical dendrites. Immunoreactivitywas particularly abundant in large pyramidal cells of the neocortex andhippocampus. Punctate staining of dendritic arborizations in themolecular layer of the neocortex suggested additional immunolabeling ofsynapses. Axons and glial cells including reactive astrocytes wererelatively weakly stained.

Affinity purified antibodies to the HMWC I-2 (41) or I-2 (87), detectedstriking abnormalities in the distribution of this antigen inneocortical and hippocampal neurons in Alzheimer's disease (AD). Incomparison to control cases where dendrites of pyramidal cells in layersIII and V were darkly stained and could be traced long distances towardthe pial surface, levels of immunoreactivity in dendrites of the sameneurons in Alzheimer's disease were markedly decreased. Perikarya ofneurons in layer III also displayed reduced levels of calpastatinimunoreactivity. By contrast, immunoreactivity in deeper layers of thecortex (IV-VI) was relatively well preserved in AD brains. Thisdistribution pattern therefore resulted in a band of dark immunostainingin layers IV-VI of Alzheimer tissue that could be differentiated fromthe uniform immunostaining of layers I-VI in control brains. Thesedifferences between control and Alzheimer patterns could be seen withoutthe aid of a microscope in many cases. A survey of 12 Alzheimer and 10control brains documented the reproducibility of these differences anddemonstrated that the Alzheimer pattern of immunoreactivity appears tobe present in the majority of AD brains that were suitable forimmunocytochemistry. The abnormality was widespread in sections ofprefrontal cortex sections and appeared to affect the dendrites of mostneurons with arborizations projecting through layers I-III. In thehippocampus, similar reductions in the level of immunoreactivity tocalpastatin were also observed in AD brains. No changes were detected inthe cerebellum. In a series of 10 brains of individuals with Down'ssyndrome (age 31-70), a pattern similar to that in Alzheimer brains wasseen in the majority of cases.

VIII. SUMMARY

These findings provide evidence for a potential role of calpastatins inthe pathogenesis of AD. Reduced levels of calpastatin in dendrites wouldprovide an explanation for observed abnormal activation of calpains inAlzheimer brains. These changes would be expected to contribute toand/or mediate the synaptic dysfunction and degeneration of neurons andtheir processes in Alzheimer's disease. The immunocytochemical patternsseen in Alzheimer and Down's brains were distinct from otherhistopathological markers of the disease process (e.g., senile plaquesand neurofibrillary tangles) and may therefore represent a usefuldiagnostic marker for Alzheimer's disease. Furthermore, restoration ofcalpastatin levels in neurons, or the pharmacologic equivalent, mayprove to be a means for reducing the rate of neuronal degeneration anddegree of synaptic dysfunction in Alzheimer's disease and thereby may bean effective treatment strategy in this disease.

Modifications of the above-described modes for carrying out theinvention that are obvious to persons of skill in medicine, immunology,hybridoma technology, pharmacology, and/or related fields are intendedto be within the scope of the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       XaaMetProProGluProAlaThrLeuLysGlyXaaValProAspAsp                              151015                                                                        AlaValGlu                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: both                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       XaaGluLysGluThrLysGluGluGlyLysProLysGlnGlnGlnXaa                              151015                                                                        XaaLysGluLys                                                                  20                                                                            __________________________________________________________________________

What is claimed is:
 1. An antibody specifically immunoreactive with animmunogen, wherein said immunogen consists essentially of asubstantially purified, low molecular weight calpastatin with amolecular weight of about 60 kilodaltons, as determined by gelfiltration, and consisting of a dimer of about 31 kilodalton subunits,as determined by SDS PAGE under reducing conditions, and wherein saidcalpastatin has a pI range of 4.2-4.7 on an isoelectric focusing gel andan N-terminal amino acid sequence as follows:NH₂-X-Glu-Lys-Glu-Thr-Lys-Glu-Glu-Gly-Lys-Pro-Lys-Gln-Gln-Gln-X-X-Lys-Glu-Lys(SEQ ID NO: 2)wherein X represents an unknown amino acid residue;wherein said calpastatin has the following amino acid composition:

    ______________________________________                                        Mole %                                                                        ______________________________________                                                Asx   8.4                                                                     Thr   3.6                                                                     Ser   10.3                                                                    Glx   17.1                                                                    Pro   5                                                                       Gly   16.4                                                                    Ala   6.4                                                                     Cys/2 0                                                                       Val   4.3                                                                     Met   0.9                                                                     Ile   3.3                                                                     Leu   3.1                                                                     Tyr   2.7                                                                     Phe   7                                                                       Lys   1.3                                                                     His   4                                                               ______________________________________                                    

wherein: Asx=sum of aspartic acid (D) and asparagine (N), and Glx=sum ofglutamic acid (E) and glutamine (Q);and wherein said calpastatin iscapable of inhibiting calcium-activated neutral proteinase activity. 2.A method of detecting pathological structures in the neurons of patientssuffering from Alzheimer's Disease and related neurofibrillarydisorders, or neurodegenerative states characterized by celldegeneration and cell death which comprises immunocytochemicallystaining said pathological structures with an antibody of claim 1.